PROJECT SUMMARY (See instructions); The goal of Project 3 is to provide high-resolution structural information that will: (1) further our understanding ofthe mechanism by which allosteric modulators regulate GPCR function, (2) form the basis for docking experiments to identify new subtype selective allosteric modulators, and (3) provide structural information for optimizing docking hits. The X-ray structures of GPCRs such as the P2AR (in both inactive and active states), the muscarinic M2, and M3, and the p- and 5-opioid receptors have enabled this project. These targets were initially selected for structural studies in the Kobilka lab based on their interesting pharmacological properties: the P2AR, an established model system for GPCR signaling; the muscarinic receptors, model systems for allosteric regulation; and the opioid receptors, which have been shown to function as homo- and hetero-oligomers, and for which there is a wealth of both peptide and small molecule ligands. The existing structures will form the basis for initial docking studies proposed in Project 1. We will work with Project 1, Project 2, and the Medicinal Chemistry core to identify and optimize novel ligands through iterative rounds of crystallography and chemical optimization. Finally, the Kobilka lab will work with the Sunahara (Project 2) and Shoichet (Project 1) labs to correlate chemical structure and functional properties with structural changes and protein dynamics determined by biochemical and biophysical approaches. Specific Aims: Aim 1. Determine active-state structures ofthe M2 and M3 muscarinic receptors. Aim 2. Obtain structures of receptors bound to positive and negative allosteric modulators. Aim 3. Determine structures of intermediate conformational states ofthe B2AR. Aim 4. Determine the structure of ligands bound to the B-opioid receptor oligomerization interface. Aim 5. Characterize the effect of allosteric modulators on receptor structure and dynamics.